The Effect Of Activation Induced Cytidine Deaminase Phosphorylation And Herpes Virus Uracil Dna Glycosylase On Antibody Diversification

Author

Marc Macaluso, The University of Texas Graduate School of Biomedical Sciences at HoustonFollow

Date of Graduation

5-2015

Document Type

Dissertation (PhD)

Program Affiliation

Molecular Carcinogenesis

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Kevin M. McBride, Ph.D

Committee Member

Rick A. Finch, Ph.D.

Committee Member

Ellen R. Richie, Ph.D.

Committee Member

Richard D. Wood, Ph.D.

Committee Member

Mark T. Bedford, Ph.D.

Abstract

Activation-induced cytidine deaminase (AID) is a mutagenic enzyme that is expressed in mammalian B-cells and initiates the antibody diversification processes of somatic hypermuntation (SHM) and isotype class switch recombination (CSR). AID is targeted to the immunoglobulin gene locus where it deaminates cytosines to generate uracil residues in DNA. This generates guanine-uracil (U:G) mismatch lesion which are recognized by uracil DNA glycosylase (UNG), a DNA repair enzyme that removes uracil from DNA and triggers downstream repair of the lesion. While UNG is a ubiquitously expressed DNA repair enzyme, its recognition and removal of AID introduced uracils is essential in both SHM and CSR. Mice lacking AID or UNG are deficient in SHM and CSR resulting in a compromised immune system.

Due to its mutagenic activity AID is heavily regulated by diverse mechanisms including post-translational modification. Here we investigate the effect of AID phosphorylation in CSR, and identify potential phospho-AID interacting factors. Using an in vitro CSR assay we show that preventing AID phosphorylation at novel sites threonine-150, serine-169 and tyrosine-184 significantly reduces CSR efficiency. We identified potential phospho-AID binding proteins using a protein domain array screen, which may prove to be important in modulating AID activity.

Herpes viruses posses a large double stranded DNA genome and many carry a form of uracil DNA glycosylase. Many of these viruses, such as Epstein-Barr virus (EBV), infect mammalian B-cells. Our experiments show that viral UNGs are able to partially restore CSR when expressed in B-cells from Ung deficient mice. Intriguingly we discovered that viral UNGs suppress CSR when expressed in wild-type B-cells, and that UNG catalytic activity is not required for this suppressive effect. We also found that viral UNG expression in mouse B-cells reduces the frequency of AID induced IgH-cMYC translocations, suggesting that viral UNG may play a role in protecting the host cell genome from damage. These studies have revealed a novel mechanism by which herpes viruses might impede the host immune system.